High Summer Temperatures Have No Role in Breaking Physical Dormancy Among Seeds of Fireprone Cistaceae: A Reinterpretation of Luna (2020)

In a unique study, Luna (2020) examined the viability and germination of 12 hard-seeded Cistaceae in the Mediterranean Basin by alternating a prolonged summer-type-temperature (50/20°C at 12 h cycles) treatment with a re-type heat pulse. A re-analysis of their data shows that the summer treatment applied before the heat pulse was superuous as similar high levels of germination under ambient conditions were attained with the heat pulse only. The abundance of hard seeds remaining when the summer treatment was applied after the heat pulse is better explained by ungerminated seeds having become hard again rather than not responding, i.e., showing secondary physical dormancy, and thus became ‘desensitized’ to their environment. While this response is adaptive, such a retarding effect will be limited in practice as most res are expected in autumn, at least historically, and are thus close to the start of optimal winter conditions for germination. Future studies should concentrate on the fate of the water-gap plug during such alternating treatments and also ensure that realistic summer temperature regimes are used.


Introduction
There has been recent interest in conditions that promote germination of 'hard' seeds (that do not imbibe water when soaked at ambient temperatures) with a focus on the family Cistaceae. Santana et al. (2020) concluded that climate and not re-proneness was the key to understanding the evolution of Mediterranean species in this family, giving high summer temperatures as more signi cant than re heat in stimulating germination. Much of the argument for a major role of high summer temperatures in promoting germination of the Cistaceae arises from experiments among other taxa that show application of temperatures above ambient but below those associated with re, such as 50/20°C for one month, give levels of germination above the controls (Ooi et al. 2014, Cochrane 2017. Such temperatures can be reached (brie y) by soil surfaces over summer in mediterranean climates (Brits 1986, Moreira and Pausas 2012). However, Moreira and Pausas (2012) showed re-type temperatures caused much greater germination of hard-seeded Cistaceae than summer-type temperatures. Here, the greater levels of response to the higher temperatures associated with re are simply viewed as exaptations (Lamont and He 2017) by the climate advocates.
The possibility of a synergistic effect between high summer temperatures and re on eight Cistus species and four Halimium species (Cistaceae) has recently been examined by Luna (2020). They exposed these species to 1−2 months at 50°C continuously or 50/20°C at 12-h cycles and compared the results against dry heat at 100°C for 10 min. All pretreatments were followed by incubation at 20°C. In addition, Luna alternated the 'summer' and heat pretreatments to see if this had any differential effect. They concluded that high summer temperatures primed ('sensitized') the seeds for maximum germination in the presence of re such that they "work together" to break physical dormancy. Submission to a heat pulse before summer failed to do that -many seeds were shown to be hard after incubation. Cistaceae are able to imbibe water once the 'water gap' (chalazal oculus) opens in response to heat (Gama-Arachchige et al. 2013). We cross-referenced the data Luna (2020) provided and performed separate analyses with a view to seeking an alternative explanation -that re heat was su cient in itself to break dormancy and that any ungerminated seeds present had in fact became hard again over the month of post re high temperatures (as shown possible for Trifolium subterraneum, Hagon and Ballard 1970). We further examined the likelihood that res occur early rather than late in the reprone season so providing little opportunity for post re, high summer temperatures to reduce subsequent winter germination. We also comment on what can be considered realistic summer temperatures to apply to hard seeds in future studies.

Methods
Germination data for eight Cistus species and four Halimium species (Cistaceae) were selected from the Supporting Material in Luna (2020). They were subjected to one or two-way analyses of variance using Vassarstats.net (©Richard Lowry, 1998-2020). Soil temperature data were collated from Brits (1986).
The literature was searched for data on a) where Cistaceae seeds are stored in space, b) likely temperatures reached by soil-stored seeds over the year, and c) monthly values of variables likely to affect the incidence of re and conditions suitable for germination in the W Mediterranean Basin. These data were collated to identify the time of year when: a) the vegetation is most vulnerable to ignition, and b) conditions are optimal for germination.

Results And Discussion
Luna (2020) noted that most ungerminated seeds that had been given a heat treatment after one month at 50/20°C were soft (they imbibed water when immersed) in contrast to those that were heated before the summer treatment. As a result, they suggested that summer temperatures prime ('sensitize') the seeds to soften more readily in the presence of re-type heat and thus serve to promote germination. The grounds for the sensitizing effect by the summer pretreatment was the heat-then-summer treatment gave 32.6 % germination and the summer-then-heat treatment 80.7 % germination (P < 0.0001, paired t-test, recalculated from Table S4 in Luna 2020). However, this pairwise comparison confounds two treatments given simultaneously: a summer treatment at time 1 and 2, and a heat treatment at time 2 and 1 that lacks controls. Other treatments (undertaken under the same conditions with the same batches of seeds but considered apart by Luna) included a control, summer treatment, heat treatment, and a summer-thenheat treatment (Table 1). Together, these show that germination was negligible in both the control and summer treatment, i.e., high summer temperatures did not promote germination. Fire-type heat caused a 7−8 times increase in germination, whether or not the seeds received a summer treatment. That is, the seeds did not need to be 'sensitized' to germinate fully in response to the heat pulse. A similar comparison can be made by replacing the heat-then-summer treatment by the summer-then-heat treatment (Table 2). Here, both sets of heated seeds germinated much more than the unheated (72% of variance), but it is the interaction effect that is of most interest: the heat-then-summer sequence yielded 55% fewer germinated seeds than the summer-then-heat sequence. Luna (2020) interpreted this as failure of most of the heat-then-summer-treated seeds to be softened by the heat in contrast to the summer-then-heat-treated. But Table 1 makes it clear that summer priming was not required in the presence of the same heat treatment. Fluctuating dormancy has also been recorded among other Cistaceae: Fumana ericoides seeds were shown to go from soft to hard and soft again over time (Llorens et al. 2008). Most Cistus clusii seeds from two populations did not germinate after storing them air dry at 4°C, in contrast to those kept at room temperature (Castro and Romero-García 1999). Most Helianthemum salicifolium seeds would no longer germinate after storing under "cold, dry" conditions in contrast to those kept at room temperature (Sánchez et al. 2014). We interpret both these cases as drying having imposed physical dormancy but note that Castro and Romero-García (1999) thought it was secondary physiological dormancy without checking the seeds for hardness nor appreciating that cold strati cation requires moist conditions. Both these cases are consistent with the dry summer treatment of Luna (2020) 'desensitizing' some seeds by imposing dormancy on them ( Table 2).
If low humidity is the key, then a month at low humidity might be as effective in restoring physical dormancy in Cistaceae as the 50/20°C used here. The heat-then-summer-treated seeds did remain viable according to Luna's 'cut' test and so these would be available to germinate following a subsequent re. Thus, the response can be considered adaptive. This also can explain the few soft ungerminated seeds among those in the summer-then-heat-treated seeds: they had not yet had time to harden again.
Assuming that the cut test is unreliable under these circumstances, it is also possible that the seeds which failed to germinate had actually died through the additional desiccation that would now occur if the pore remained unplugged over the extended period at 50°C. [One of us, GB, observed considerable additional water loss by Acacia seeds once the lens had popped though they did not die nor become hard again]. We agree with Luna that the relative location of the pore and plug needs to be examined by electron microscopy after various drying/heating treatments to resolve these alternative interpretations (personal communication). Bastida and Talavera (2002) showed that most seeds of two Cistus species were stored beneath the plant crown. Tuberaria guttata seeds are stored < 10 cm from the parent plant (Guarino, Ferrario and Mossa 2005). Further, Cistaceae seeds are tiny (< 2 mg) and spheroid (Bastida et al. 2009) so will easily penetrate the litter to reach the soil. These observations indicate that invariably Cistaceae seeds will be stored in litter-rich parts of the soil surface. Seeds of some species are dispersed by harvester ants but these are unlikely to account for more than 20% of their nal location (Bastida et al. 2009). Some seeds can be ingested without harm by ruminants -here the seeds remain in the dung and are not exposed to What temperatures then are reached at soil locations where Cistaceae seeds are likely to be stored? Brits (1986) studied the likely temperature uctuations in pre-and post-burn surface soil in detail. For a site matching parts of the Mediterranean Basin, this work showed for the hottest month of the year that, at a soil depth of 5 mm, the warmest 12-h period of the day averaged 41°C and the coolest 22°C under light shade, and 28°C and 22°C under heavy shade (Table 3, see Moreira and Pausas 2012 for similar results).
Thus, a summer pretreatment temperature at 50°C continuously for 1−2 months (Luna 2020) appears extremely high compared with expected summer temperatures at a depth of 5 mm even for mineral soil exposed after re (47/28°C, Table 3), while that at 50/20°C 12-h cycles seems a reasonable simulation of unburnt sites under light shade. However, the data reviewed above indicate that heavily shaded conditions at greater (cooler) depths are more likely for stored Cistaceae seeds in the Mediterranean Basin. Table 3 Mean daily temperatures (to nearest 0.5°C) at a depth of 5 mm for the warmest month of the year at the ¾ position (midway between the mean and maximum for the warmest 12 h) and ¼ position (midway between mean and minimum for the coolest 12 h) of the temperature range for three possible locations in shrubland. Compiled from Brits (1986) for a mild Mediterranean-type climate in South Africa. These provide a guide as to suitable temperatures to set on a 12/12-h cycle if attempting to simulate temperature conditions at the hottest time of year where stored seeds are located at 5 mm depth.
Part of temperature range ). This is supported by the abrupt but broad peak in re 'danger' days at this time (Dimitrakopoulos et al. 2011) (Fig. 1). The moisture content of living ne foliage of Cistus species is used as an index of re-proneness (Pellizzaro et al. 2007, Chuvieco et al. 2009) and low levels cover this period. Mean air temperatures also peak during the re season. Monthly rainfall is at a minimum but does rise sharply in September, at least on the E side of the Iberian Peninsula (the source of Cistaceae studied by Luna 2020). As a suitable ignition source, the incidence of lightning shows that there is a sharp peak in September to December (Rivas Soriano and de Pablo 2002, Price and Federmesser 2006). At a depth of 5 mm, means of the monthly soil temperatures at the extremes of post re bare ground and pre re dense shade peak in July (Brits (1986). We note that the deep-shade pattern is almost the same as that for the Stevenson-screen air temperatures for Valencia (www.climatestotravel.com/climate/spain/valencia) and indicates this could be used as a reasonable surrogate for shaded surface soils.
Putting these data together shows that, when the re regime is controlled by lightning, res are most likely to occur at the end of the reprone season, September (Fig. 1). This is about six weeks away (October-November) from soil conditions optimal for germination of Cistaceae seeds ( . Thus, the period between breaking dormancy and germination opportunity is short, soil temperatures are 7°C less than the summer peak, and autumn rains are already substantial. This means that any return to dormancy as suggested here during the remaining post re, summer-autumn period will be short so that it has little role in reducing the extent to which previously heated seeds will germinate. More important will be the type of re (He, Lamont and Pausas 2019) and properties of the wet season (Céspedes et al. 2012). We agree with Luna (personal communication) that seed burial under different eld conditions is required to ensure future studies mimic what occurs in nature.
In conclusion, the simulated summer treatments need to be compatible with where seeds are actually stored over summer. It is clear that a continuous 50°C for 1−2 months has no equivalent in nature and its only use might be in the context of exploring pre-adaptations (Lamont and He 2017). Most buried seeds will be well insulated from sunlight and mean temperatures in the order of 20-40°C, as obtained under shade over the hottest summer month under a mediterranean climate by Brits (1986), appear more reasonable. Any differential effect of varying the sequence of heat and 'summer' treatments is of ecological and management relevance. If the interest is in the effect of long periods of severe summer heat following re on seed viability and germination, then a re-type heat treatment followed by 1−2 months at 50/20°C realistically simulates conditions that Brits (1986) determined for post re bare areas at a depth of 5 mm (Table 3). It is well established that late season res result in greater seedling establishment than early season res (Enright and Lamont 1989). The usual explanation is in terms of greater dormancy-breaking e ciency (or seed release by serotinous species) by more intense res rather than early res resulting in a return to dormancy before winter rains begin as our preferred interpretation of Luna's data. Only studies that overtly studied the presence of dormancy cycling would be in a position to distinguish these two possibilities. The results could be added to guidelines on the most suitable time for management res, bearing in mind that Enright and Lamont (1989) showed that recruitment success of early and late res tended to merge with time.